microRNAs (miRNAs) are short, non-coding RNA molecules, which are endogenously expressed either ubiquitously or in a tissue-specific manner, and play an important regulatory role in various cellular processes. miRNAs are of about 21 nucleotides in length that are part of a mechanism that regulate posttranscriptional gene expression. miRNAs are expressed in organisms as diverse as nematodes, fruit flies, humans and plants. In mammals, miRNAs are generally transcribed by RNA polymerase II and the resulting primary transcripts (pri-miRNAs) contain local stem-loop structures that are cleaved in the nucleus by a Drosha-DGCR8 complex. The product of this cleavage is one or more (in case of clusters) precursor miRNA (pre-miRNA). Pre-miRNAs are usually 70-90 nucleotides long with a strong stem-loop structure containing a 2 nucleotides overhang at the 3′ end. The pre-miRNA is transported to the cytoplasm by Exportin-5. In the cytoplasm, the Dicer enzyme, which is an endoribonuclease of the RNase III family, further cleaves the pre-miRNA to release a 21 bp dsRNA, the miRNA duplex. The two strands of the duplex are separated from each other by the Dicer-TRBP complex and the strand that usually has thermodynamically weaker 5′ end is incorporated into the RNA induced silencing complex (RISC). This strand (guide strand) is the mature miRNA. The strand which is not incorporated into RISC is called miRNA* strand (3p strand or passenger strand) and it is degraded, but occasionally both strands function as mature miRNAs. The mature miRNA guides RISC to a target site within mRNAs. If the target site has perfect complementarity to the mature miRNA, the mRNA is cleaved at a position that is located about 10 nucleotides upstream from the 3′ end of the target site. After the cleavage, the RISC-mature miRNA strand complex is recycled for another activity. If the target site has lower complementarity to the mature miRNA, the mRNA will not be cleaved at the target site but the translation of the mRNA will be suppressed.
miRNA-122 is the most abundant liver specific microRNA, representing 70% of total liver miRNAs. miR-122 was recognized as a tumor suppressor miRNA. Its expression commences during gestation and attains maximal levels in the adult liver. miRNA122 plays a fundamental role in cholesterol and fatty acid metabolism. It has been shown to be required for the replication of hepatitis C virus (HCV). miR-122 was shown to be either silent or expressed at a very low level in most hepatocellular carcinomas (HCCs). U.S. Pat. No. 7,232,806 is directed to microRNA molecules and discloses miR-122 and complementary oligonucleotides thereof.
The discovery of RNA interference (siRNA) has enabled selective and highly efficient targeting of specific genes, including genes harbored by disease causing agents. RNA interference entails the introduction of short, double stranded RNA (termed small interfering RNA-siRNA) into cells which results in degradation of defined homologous target gene. Both microRNAs and siRNAs share similar machinery for recognition of their target genes.
Human papilloma viruses (HPVs) are small DNA viruses, which have been linked to a variety of epithelial cancers. Despite the introduction of screening with cervical cytologic testing (Pap test), cervical cancer remains the second most common cancer in women worldwide. Over 120 types of HPV have been identified and classified according to their tropism. A sexually transmitted subgroup consisting of several HPV types is categorized as high-risk HPVs that are responsible for over 99% of virus-mediated cervical lesions. The most prevalent high-risk types of HPV are HPV16 and HPV18, accounting for 50% and 20% of all cervical cancers, respectively.
The HPV life cycle initiates when a virus infects immature cells from the basal layers of stratified epithelia. These basal cells become exposed as a result of micro-wounds and are the only proliferating cells in normal epithelia. The HPV oncoproteins E6 and E7 are the primary viral factors responsible for initiation and progression of cervical cancer, by cooperatively acting to overcome the host cell's natural defense mechanism against uncontrolled cell division. The primary target of E7 is the Rb (retinoblastoma) tumor suppressor protein, one of the key regulators of the cell cycle. The efficient abrogation of Rb function by E7 leads to increased levels of the tumor suppressor p53, which induces the susceptibility of E7-expressing cells to apoptosis. The E6 proteins interfere with p53, abolishing its function, either by direct binding or by recruitment of cellular mechanisms for protein degradation to avoid the cellular innate response. The combined expression of E6 and E7 proteins immortalizes most types of primary cells.
Currently, pre-malignant cervical lesions are treated by local excision. This procedure has a number of disadvantages: performed only on visible lesions, destroys cervical tissue and it is associated with high recurrence rates. Advanced cancers are treated by a combination of chemotherapy and radiotherapy that have serious adverse effects.
Therefore, there is a need in the art for selective, efficient and safe treatment of various types of pre-malignant or malignant conditions. In particular, there is a need in the art for selective, efficient and safe treatment of various conditions such as, for example, HPV associated pre-malignant and malignant lesions.